The overall objective of this proposal is to develop more effective diagnostic approaches to human breast cancer using monoclonal antibodies (MAbs) reactive with breast carcinoma-associated antigens. We have prepared a murine MAb, designated DF3, against a membrane-enriched fraction of a human breast carcinoma metastatic to liver. Immunoperoxidase staining has demonstrated the presence of DF3 antigen in the cytosol of malignant breast epithelial cells. The expression of DF3 antigen has also been shown to correlate with the degree of breast tumor differentiation and estrogen receptor status. Furthermore, DF3 antigen is produced as heterogeneous species with molecular weights ranging from 300 to 400 kd. Studies in family members indicate that the electrophoretic heterogeneity of DF3 antigen is determined by codominant expression of multiple alleles at a single locus. Double-determinant immunoassays developed to monitor circulating DF3 antigen have demonstrated that changes in DF3 values correlate with clinical course in patients with metastic breast cancer. These findings have resulted in the development of a bideterminant immunoradiometric assay which utilizes both MAb DF3 and another MAb, designated 115-D8, which reacts with a different epitope on this antigen. This assay, which detects the CA15-3 antigen, has recently been shown to be more sensitive than the currently used carcinoembryonic antigen assay for the evaluation and monitoring of patients with breast cancer. The proposed work represents an extension of studies initiated during the current funding period. We will continue characterization of the DF3 antigen with respect to the core protein, carbohydrate structure and polymorphic expression (Specific Aim 1). Immunoperoxidase staining studies will examine DF3 antigen expression in breast tumor and lymph nodes (Specific Aim 2). Prospective clinical trials will evaluate the utility of monitoring CA15-3 levels in patients with breast cancer (Specific Aim 3). Furthermore, we will determine the usefulness of MAb DF3 in the radioimmunodetection of breast cancer metastatic to axillary lymph nodes (Specific Aim 4). These studies should be important in providing the basis for using MAb DF3 in the immunodiagnosis of human breast cancer.